The type II secretion pathway is widely distributed among Gram-negative pathogens where it is responsible for extracellular secretion of toxins and degradative enzymes. At least 12 gene products located in both the cytoplasmic and outer membrane collectively comprise the type II secretion apparatus, which is specifically required for the translocation of secreted proteins from the periplasm to the extracellular milieu. This pathway is highly specific. It distinguishes secreted proteins from resident periplasmic proteins and discriminates between its own secreted proteins and those introduced from closely related species. In addition, secretion through this pathway differs from most other membrane transport systems in that its substrates consist of folded proteins. The objective of this proposal is to characterize the mechanism of type II secretion at the molecular level. The studies will utilize Vibrio cholerae and will specifically examine the secretion of cholera toxin (CT), as well as other proteins that utilize the type II pathway. CT provides an excellent model protein for these studies since its structure and function as well as its folding and assembly pathway are very well characterized. Specifically, we will test the hypotheses that: 1) Molecules secreted by the type II system encode information critical to their secretion within their tertiary structure, 2) the type II complex spans both membranes and components involved in connecting the outer membrane secretion pore with the cytoplasmic membrane regulate secretion by transducing energy to the secretion pore or by regulating its opening, 3) the secretion apparatus is localized to the poles or septum where the peptidoglycan undergoes rearrangements and/or where the pore size is atypical to allow for secretion of folded proteins.